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Toxic Insult to Rat Precision Cut Lung Slices Increases Tissue Cytokine Levels and Activation of Macrophages, and Causes Acute Damage, While Prolonged Insult May Lead to Increased Deposition of Collagen - a Marker of Fibrosis
The use of in vitro or ex vivo models is intended to provide meaningful data that will identify or predict the adverse effects of tissue exposure. Precision-cut lung slices (PCLS) are used as a model that retains the heterogenous population of cells in the native architecture of the organ. The retention of native cells allows the study of the initial, dynamic events (such as inflammation) that occur following a toxic insult prior to overt tissue damage. The purpose of the reported studies was to identify initial inflammatory signals, acute toxicities, as well as markers associated with chronic toxicities of PCLS exposed to a toxic insult as a way to qualify the model for identifying such endpoints. Rat PCLS were exposed to several chemotherapeutics known to cause acute and/or chronic pulmonary damage. Time points for respective endpoints were chosen based on known response times of when relevant endpoints may change. Cytokines and acute toxicity were evaluated during initial days of exposure while activation of macrophages and collagen deposition were evaluated through 4 weeks of culture in other studies. Exposure of PCLS for 24 hours resulted in increased cytokine levels and 72 hour exposure caused overt toxicity, as assessed using tissue protein content and histologically using H&E and ED-1 staining. Long term exposure of PCLS to two agents known to cause fibrosis (bleomycin and carmustine) resulted in elevated numbers of macrophages and also increased collagen deposition. PCLS generate inflammatory cytokine signals and, if levels persist after insult removal, these signals may predict subsequent tissue damage. The expression of adverse markers of chronic exposures (collagen deposition) in PCLS may signify risk of fibrosis. Cytokine responses, macrophage activation, and fibrosis are hallmarks of tobacco related exposures. PCLS may elucidate acute and chronic adverse pulmonary responses when exposed to tobacco products.
Evaluation of a Reconstructed Human Oral Buccal Tissue Model as a Testing Platform for Determining the Oral Irritation Potential of Tobacco Products
There is an increasing need by the tobacco industry to evaluate the irritation and inflammation potential of tobacco products to support product development goals, for sound product stewardship, and likely regulatory safety tests. The use of in vitro human cell and tissue-based test methods to replace in vivo animal models addresses the need for more human-relevant predictive tools, and is consistent with many corporate animal welfare policies. Although monolayer cell-based cytotoxicity and cytokine expression assays have been used, three-dimensional tissue constructs provide distinct advantages since tissue exposures and pharmacokinetics more closely resemble the in vivo events. In this study, we evaluated a reconstructed human oral buccal model for determining the oral irritation of oral tobacco products. A dilution series of tobacco extracts were applied topically onto the EpiOral™ reconstructed human oral buccal model (Cat no. ORL-200) (MatTek Corporation, Ashland, MA) for various exposure times (ranging from 2 to 16 hours) to estimate oral irritation potential based upon reduction in cell viability and the synthesis/release of the inflammatory mediators IL-1α and IL-8. We determined tobacco extract concentration-related increases in cytotoxicity for the highest tobacco extract concentrations. We also found that increases in IL-1α release (up to 19-fold) generally correlated with the cytotoxicity increases. Exposure time related increases in IL-8 release were generally observed in tissues treated with the three lower tobacco extract concentrations where relative viabilities were sufficiently high to allow for secondary cytokine production, but at the highest tobacco extract concentrations IL-8 release were below control levels where cytotoxic effects inhibited the cells’ ability to synthesize proteins. These results demonstrate the utility of reconstructed human epithelial models for evaluating the irritation potential of tobacco products. To expand upon this utility, we propose to apply these general methods for determining cytotoxicity and inflammatory cytokine profiles to evaluating inflammation responses in reconstructed human airway tissue models exposed to combustible tobacco product extracts, particulate matter, and whole smoke.
Preliminary Investigation on Reducing Ocular Irritation Potential of Harsh Ingredients By Increasing Formulation Viscosity
Formulations tested for ocular irritation using the Bovine Corneal Opacity and Permeability (BCOP) assay may be assigned a specific irritation label based on the resulting In Vitro Irritancy Score (IVIS) and specific regulatory guidelines (e.g. OECD, EPA, and CLP) that provide cutoff values for classifications. The ability to reduce ocular irritation by slightly adjusting the physical properties of a formulation is highly desirable. Laboratory investigations found that incrementally changing viscosity using increasing amounts of Carbopol® as a thickening agent reduced ocular irritation when mixed with a 1% NaOH solution in water. Following a 10-minute exposure in the BCOP assay, 1% NaOH was previously classified as a severe ocular irritant (IVIS=161.6). Increasing Carbopol® from 0.25% to 1.25% in a mixture with 1% NaOH decreased the In Vitro score to a range of values between 150.9 and 18.3 and decreased ocular irritation across a range of irritation classifications from severe to mild irritation (n= 3 corneas per treatment). Exposure to 1% Carbopol® alone exhibits minimal irritation (IVIS=1.3) and Carbopol® is consequently not considered to contribute to ocular irritation within the tested mixtures. Histopathology evaluation further supports that exposure to 1% Carbopol® results in damage similar to negative control treated corneas and that epithelial and stromal damage decreases as viscosity increases. Additionally, preliminary findings indicate that when a small amount of thickener is added to a complex formulation containing otherwise harsh ingredients, ocular irritation can be mitigated from a Category I label to a Category II label according to current EPA guidelines applicable to cleaning products making anti-microbial claims. Similarly, increasing the viscosity of a formulation containing more than 3% of a severe ingredient also resulted in a “Not Classified” label according to OECD criteria when it would have received a severe classification if left untested (according to CLP regulations). These results indicate that increasing viscosity may be an effective tool for reducing ocular irritation potential of a formulation. Viscosity, among other physical properties, may therefore be used to inform decision making during product development, ultimately affecting downstream users in such areas as marketing, labeling, packaging and distribution.
Several in vitro eye irritation models exist; however, no eye irritation models have demonstrated the ability to accurately predict eye stinging. The NociOcular assay, a novel neuronal model based on activation of the Transient Receptor Potential Vanilloid type 1 (TRPV1) channels, has been shown to distinguish stinging from non-stinging products. In the NociOcular assay, the TRPV1 channel expressing SH-SY5Y neuroblastoma cells are exposed to a serially-diluted test substance and TRPV1 channel activation is measured by acute increases in the intracellular free Ca2+. Although the NociOcular assay was originally designed to predict the eye sting potential of surfactant ingredients and surfactant-based products, there are many other product types which may come in contact with the eyes, such as sunscreens. In this study, we sought to evaluate sunscreens and other products that are used near the eyes. We developed alternate solvents and a modified dilution method to overcome solubility and viscosity limitations and to more accurately model in-use exposures. Furthermore, we investigated the possibility that the alternate solvents and modified dilution method could affect the results of the assay. During proof-of-concept studies, the assay modifications allowed for greater solubility, and controls performed as expected. Additionally, using these modifications of the assay, we successfully measured TRPV1 channel activation caused by products which are hydrophobic, viscous, and may come into contact with the eyes at a high concentration. Future research will focus on evaluation of target ingredients in insoluble products and further modifications of the assay to assess final product formulations.
Assessing Increased Sensitivity and Variability Issues in an Established In Vitro Phototoxicity Testing Program
The 3T3 Neutral Red Uptake (NRU) Phototoxicity assay is an established in vitro assay used to evaluate the potential phototoxicity hazard of a test chemical. The assay methods and prediction model are described in The Organization for Economic Cooperation and Development (OECD) Test Guideline (TG) 432 “In Vitro 3T3 NRU phototoxicity test”1. IIVS’ routine assay performance is evaluated on the comparison of the positive control, chlorpromazine, and solvent control results to our historical database. Failure to consistently meet acceptance criteria calls for examination of assay performance. High assay sensitivity and low optical density values have contributed to the failure of recent assay results to meet acceptance criteria. Storage conditions, preparation, and manufacturer lot-to-lot consistency of assay reagents (DMSO, chlorpromazine, and neutral red) were evaluated. Variations in positive control or solvent control responses in different DMSO (lot-lot or catalog number). Preparation, storage condition, and filtration methods affected neutral red signal. UVA light source investigated for impacts on irradiance uniformity. Variations in UVA light intensity observed depending on plate placement under the light source. Several variables (reagents and light source) which likely impacted assay performance and may have contributed to increasing assay sensitivity were investigated.
The Use of Precision-cut Lung Slices to Assess Inflammation, Parenchymal Damage, and Collagen Deposition: Three Markers of Tobacco Exposure-induced Pulmonary Toxicity
The Family Smoking Prevention and Tobacco Control Act (TCA) gives the Food and Drug Administration (FDA) broad authority to regulate tobacco products. The FDA Center for Tobacco Products (CTP) calls for several research priority areas, including in vitro models and assays that will assess tobacco constituent safety and comparative product toxicity. Precision-cut lung slices (PCLS) retain normal lung architecture and endogenous cell types and have been effective in assessing acute pulmonary toxicity. Having demonstrated release of traditional leakage markers, inflammation, and tissue damage, recent advances in PCLS culture and choice of biomarkers suggest suitability for detecting specific events that contribute to the etiology of tobacco-induced chronic obstructive pulmonary disease (COPD). PCLS were exposed to various compounds known to cause acute and chronic pulmonary lesions. Acute toxicity was assessed via cytokine level, protein content, and the overt destruction of parenchymal tissue. Chronic exposures also included quantifying activated macrophages and assessing a marker of fibrosis (in addition to general histopathological evaluation). Acute toxicity elicited a rapid increase in cytokine levels, reduction in tissue leakage markers, and overt parenchymal damage. Chronic exposures lasting up to several weeks also resulted in decreased tissue viability, increased numbers (and location of) activated macrophages (ED-1 positive cells) and deposition of collagen. In summary, PCLS data from various chemical assessment studies are compiled to highlight the changes in different markers of acute and chronic toxicity, as related to the known events of tobaccoinduced COPD etiology. It is expected that PCLS will prove to be a valuable tool in elucidating both acute and chronic effects of compounds found in tobacco or modified risk tobacco products such as e-cigarettes.
Although many cosmetic, personal care, consumer product and raw material supplier companies have been working for decades to eliminate animal testing, in some countries regulatory authorities still require animals for product safety testing. While there are differing hurdles to acceptance of non-animal methods around the world, a common difficulty is lack of technical training. IIVS, a non-profit and world leader in the validation, training and application of non-animal test methods, has organized a group of companies to form the Industry Council for the Advancement of Regulatory Acceptance of Alternatives (ICARAA). ICARAA is a working group which provides counsel and financial support of IIVS’ mission to increase the use and adoption of in vitro methods internationally. Led by IIVS, ICARAA activities focus on educational programs that include lectures, hands-on training and data interpretation. Many of ICARAA’s activities are currently in China where there is keen interest on the part of the regulatory authorities to understand how non-animal approaches can be used to substantiate safety. This collaboration between regulatory agencies, industry and a technical institute serves as a model example of how to promote the practical acceptance of non-animal techniques and facilitate the movement away from animal testing for regulatory purposes.
3D reconstructed skin tissues provide a more realistic model for dermally applied chemicals/products, such as cosmetics, and are expected to be used to follow-up on positive results from the in vitro genotoxicity battery1. Phase 1 and 2 of the RSMN validation using EpiDermTM tissues showed good transferability, inter- and intralaboratory reproducibility2,3. In Phase 3, the number of chemicals was extended to 29 (Table 1). Results demonstrated excellent specificity and good within-laboratory reproducibility (Table 2 and 3), while sensitivity needs further investigation.
A TRPV1 expressing clone of the human SH-SY5Y neuroblastoma cell line (Figure 1) was obtained by stable transfection, using puromycin-containing selection medium. Prior to Ca2+ measurements the TRPV1-SH-SY5Y cells were cultured in 96-well plates. Acute increase in the intracellular free Ca2+ level was measured in a semi-HTS fluorescence reader (FlexStation, Molecular Devices) using Fura-2/AM. The ratio of fluorescence at 340 (Ca2+-bound Fura-2)/380 (Fura-2) nm excitatory wavelengths was registered without interruption before and during the 2 min exposure to the test compounds. The mean value (% increase of basal Ca2+ level) from triplicate wells in the 96-well plate was monitored for each concentration from each experiment. The TRPV1 antagonist capsazepine was added simultaneously with each concentration of the chemicals in three sister wells to confirm TRPV1-mediated Ca2+ influx. The intracellular Ca2+ increase induced by the specific TRPV1-agonist capsaicin was set to 100% response for each experiment and the effect of the test products was calculated as percent of the capsaicin induced response. All test compounds were diluted in KRH-buffer and the addition to the cells was performed robotically during measurements by the FlexStation reader.
Identifying the Appropriate Protocol for Testing Surfactants and Surfactant-based Formulations in the Bovine Corneal Opacity and Permeability Assay
The Bovine Corneal Opacity and Permeability (BCOP) assay is an ex vivo test for predicting ocular irritation. OECD Test Guideline (TG) 437 specifies that liquid and solid surfactants are tested as 10% aqueous dilutions for 10 minutes (alternate dilutions and exposure times may be conducted with scientific rationale). Guidance Document (GD) No. 160 suggests that solid and concentrated liquid surfactants may be diluted to 10% for testing, and thus surfactant solids should not be tested using the solid chemical protocol. GD No. 160 further directs that surfactant-based formulations are usually tested neat, but could be diluted with justification, imparting some confusion in identifying the most appropriate test methods. In the absence of clear guidance, we present on the testing of a few common surfactant ingredients (sodium lauryl sulfate, Triton X-100, and benzalkonium chloride), and surfactant-based liquid and solid formulations in BCOP using standard and modified dilutions and exposures to evaluate the impact of these variables. Histopathology was performed to confirm corneal changes. We found that surfactants may not exhibit dose-related effects at high concentrations, and opacity and permeability changes should be evaluated individually in a hazard assessment. Accordingly, a framework for testing surfactants and surfactant-based formulations is proposed.